Isomalto-oligosaccharides (“IMOs”) are mixed linkage oligosaccharides, having mixtures of 1,4 alpha and/or 1,6 alpha glucosidic linkages. They are also known as anomalously linked oligosaccharides (“ALOs”). Isomalto-oligosaccharides contain a substantial amount of branched oligo-saccharides such as isomaltose, panose, isomaltotriose, isomaltotetraose, isopanose and higher branched oligo-saccharides.
There is a market demand for products containing IMO's. IMO products are sold in powder or liquid form, depending on the intended application. The potential applications are situated in the food area. Examples of IMO products are: seasonings (mayonnaise, vinegar, soup base etc.), confectionery (candy, chewing gum, chocolate, ice cream, sherbet, syrup), processed foods of fruits and vegetables (jam, marmalade, fruit sauces, pickles), meat or fish foods (ham, sausage, etc.), bakery products (bread, cake, cookie, pastry), precooked foods (salad, boiled beans, etc.), canned and bottled foods, convenience foods (instant coffee, instant cake base, etc.), and beverages, both alcoholic (liquor, seju, wine, sake, beer [International Publication No. WO 02/20712 A1], etc.) and non-alcoholic (coffee, juice, nectar, aerated or carbonated drinks, lemonade, cola). Isomalto-oligosaccharide can further be applied as ingredients in animal feed and pet foods. Non-food application areas are cosmetics and medicine (cigarette, lipstick, toothpaste, internal medicine, etc.).
Isomalto-oligosaccharides belong to a group of oligosaccharides classified as functional-health food oligosaccharides (“FHFO”). Exemplary IMO's include fructo-oligosaccharides, galacto-oligosaccharides, xylo-oligosaccharides and gentio-oligosaccharides. IMO's have been linked to the increase of the general well being of humans and animals when taken orally on a regular daily basis and are classified as “prebiotics”. Prebiotics are defined as non-digestible substances (e.g., dietary fiber) that exert some biological effect on humans by selective stimulation of growth or bioactivity of beneficial microorganisms either present or therapeutically introduced to the intestine. (Przemyslaw Jan Tomasik and Piotr Tomasik. 2003 American Association of Cereal Chemists, Inc. 80(2): 113-117). The “prebiotic” action of the oligosaccharides is to increase the numbers of bifidobacteria and lactobacilli (“prebiotic”) in the large intestine and to reduce the concentration of putrifactive bacteria. Bifidobacteria are associated with some health promoting properties like the inhibition of the growth of pathogens, either by acid formation or by anti-microbial activity. They are also associated with such diverse effects as the modulation of the immune system (anti-tumor properties), the reduction of the levels of triglycerides and cholesterol, the production of vitamins (B group), the reduction of blood ammonia concentrations, the prevention of translocation, the restoration of the normal gut flora after anti-microbial therapy, the production of digestive enzymes, the reduction of antibiotic associated side effects (Kohmoto T., Fukui F., Takaku H., Machida Y., et al., Bifidobacteria Microflora, 7(2) (1988), 61-69; Kohmoto K., Tsuji K., Kaneko T., Shiota M., et al., Biosc. Biotech. Biochem., 56(6) (1992), 937-940; Kaneko T., Kohmoto T., Kikuchi H., Fukui F., et al., Nippon Nogeikagaku Kaishi, 66(8) (1992), 1211-1220, Park J-H, Jin-Young Y., Ok-Ho S., Hyun-Kyung S., et al., Kor. J. Appl. Microbiol. Biotechnol., 20(3) (1992), 237-242). Modler, H. W., 1992, “Compounds which enhance the growth of Prebiotic Bacteria”, presented at the International Roundtable on Animal Feed Biotechnology, Ottawa, Ontario, Canada.
The isomalto-oligosaccharides are synthesized by an enzyme catalyzed transglucosylation reaction using a D-glucosyltransferase (E.C. 2.4.1.24, transglucosidase, alpha-glucosidase). This enzyme catalyzes both hydrolytic and transfer reactions on incubation with alpha-D-gluco-oligosaccharides. The transfer occurs most frequently to 6-OH (hydroxyl group 6 of the glucose molecule), producing isomaltose from D-glucose, or panose from maltose. The enzyme can also transfer to the 2-OH or 3-OH of D-glucose to form kojibiose or nigerose, or back to 4-OH to reform maltose. As a result of transglucosidase reactions, the malto-oligosaccharides are converted into isomalto-oligosaccharides resulting in a class of oligosaccharides containing a higher proportion of glucose moieties linked to a primary hydroxyl group of a glucose molecule from the non-reducing end, e.g., by alpha-D-1,6 glucosidic linkages. The transglucosidase from A. niger acts only on oligosaccharides with a low degree polymerization (DP) (McCleary B. V., Gibson T. S., Carbohydrate Research 185 (1989) 147-162; Benson C. P., Kelly C. T., Fogarty W. M., J. Chem. Tech. Biotechnol., 32 (1982) 790-798; Pazur J. H., Tominaga Y., DeBrosse C. W., Jackman L. M., Carbohydrate Research, 61 (1978) 279-290). Degree of polymerization refers to the number of dextrose units. For example, a di-glucosyl molecule, for example maltose, has a DP of 2. These sugars are receiving increased attention as food additives because they help prevent dental caries (Oshima, et. al 1988. The caries inhibitory effects of gos-sugar in vitro and rat experiments. Microbial Immunol. 32, 1093-1105) and improve human intestinal microflora acting as a growth factor (prebiotic) for bifidobacteria (Komoto, et. al 1988; Effect of Isomalto-oligosaccharides on human fecal flora Bifidobacteria Micro flora 7, 61-69).
Isomalto-oligosaccharides can be obtained in different ways. For example glucose syrups at high dry solids concentration i.e. 60-80% are treated with glucoamylase resulting in the formation of isomalto-oligosaccharides mainly DP2. The high solids levels are present to force the reaction to reverse from the normal direction in favor of hydrolysis.
Grains, including wheat, barley, etc., are excellent raw materials in the commercial production of many value added functional food ingredients such as wheat flour, starch, starch hydrolysates (glucose, fructose, high maltose syrup, etc.) and wheat gluten. Syrup containing a high level of maltose is also used in many microbial fermentations as a carbon source in the production of antibiotics, pharmaceuticals, vaccines, biochemical, such as alcohol (both potable and fuel), amino acids, organic acids, etc and recently in the production of functional health-food oligosaccarides called isomalto-oligosaccharides. In a conventional process for the production of starch hydrolysate, such as maltose syrups, the insoluble granular starch is generally separated from other cellular components of wheat prior to the hydrolysis by starch liquefying and maltogenic alpha amylases enzymes. Maltose is a disaccharide consisting of two glucosyl residues linked by α 1-4 D-glucosidic linkage and is the smallest in the family of malto-oligosaccharides. It is produced on a large scale as syrup, powder and crystals in several grades of purity. Various maltose syrups are drawing considerable interest for commercial applications in brewing, baking, soft drink canning, confectionary and other food and beverage industries. Ultra pure maltose is used as an intravenous nutrient in Japan. Catalytic reduction of maltose results in maltitol, which is considered to be a low calorie sweetener. Recently, high maltose syrup has become a key raw material for industrial production of isomalto-oligosaccharides (J. K. Shetty and O. J. Lantero, 1999 “Transglucosylation of Malto-oligosaccharides.” Paper presented at 50th Starch Convention, Detmold, Germany).
In a conventional process for the production of starch hydrolysate such as high maltose syrup, the insoluble starch is separated prior to the hydrolysis by thermostable liquefying alpha amylases [EC 3.2.1.2,alpha (1,4)-glucan glucanohydrolase] derived either from Bacillus licheniformis or Bacillus stearothermophilus. Hydrolysis of the purified starch (refined) is carried out by suspending insoluble granular starch in water (30-35% dissolved solid basis [dsb]) and heated to a temperature of between 85° C. and 120° C. to solubilize the starch and making it susceptible for enzymatic hydrolysis. The liquefied starch is further processed to manufacture starch hydrolysate with different carbohydrate composition using specific maltose producing enzymes such as fungal alpha amylase (sold under the tradename CLARASE L from Genencor International, Palo Alto, Calif.) for syrup containing less than 55% maltose, β Amylase (sold under the tradename OPTIMALT BBA from Genencor International, Palo Alto, Calif.) for syrup containing maltose content between 55% and 62% and less than 1% glucose. For higher levels of maltose syrup, >62%, addition of debranching enzyme (sold under the tradename OPTIMAX L-1000 from Genencor International, Palo Alto, Calif.) in conjunction β Amylase is useful. (Faigh, J.; Duan, G.; Strohm, B. and Shetty, J. (2002) “Production of Maltose, High Maltose & Very High Maltose Syrups,” Technical Bulletin, Genencor International Inc.).
A process for converting granular starch (refined) into soluble hydrolysate by incubating with bacterial alpha amylase at a temperature below the starch gelatinization temperature (Leach et. al 1978; U.S. Pat. No. 4,113,509) and subsequent hydrolysis by beta amylase to produce high maltose syrup have been reported (Leach et. al 1975;U.S. Pat. No. 3,922,196), However the syrup produced by such process resulted in only 55% maltose of the total sugar content, with a very high level of maltotriose. The process for producing high maltose syrup using liquefied starch (gelatinized followed by hydrolysis using thermostable alpha amylase) is described in European Patent Application #0905256 (Christophersen, et. al 2000) and U.S. Pat. No. 5,141,859 (Nimmi, et. al 1992). The process is cumbersome, expensive and it requires the separation of starch from other cellular components, high cost of the additional maltose producing enzymes, high temperature treatment and longer reaction time. European Patent Application #0350737 A2 (Shinke, et. al 1989) disclosed a process for producing maltose syrup by hydrolyzing a granular (purified) starch from corn, wheat, potato and sweet potato at 60° C. without the conventional liquefaction step (gelatinization followed by liquefaction at high temperature) using an alpha amylase from Bacillus stearothermophilus. However, the hydrolyzed starch resulted in a maltose concentration ranging from 50% to 55%. The syrup also contained very high level of maltotriose (30-36%). The process resulted in a ratio of maltose to maltotriose less than 2.0 irrespective of the source of the starch. Maltose syrup containing a high level of maltotriose is not a preferred substrate as carbon feed in many microbial fermentations including the alcohol fermentation by yeast because of the difficulties in metabolizing maltotriose. Maltose is a preferred donor of glucosyl residue in the transglucosylation reaction catalyzed by glucosyltransferases in the production of isomalto-oligosaccharides (J. K. Shetty and O. J. Lantero, 1999 “Transglucosylation of Malto-oligosaccharides.” Paper presented at 50th Starch Convention, Detmold, Germany). U.S. Pat. No. 6,361,809 described a method for producing maltose and a limit dextrin by treating the purified granular waxy maize starch with a hydrolase, maltogenase alpha amylase classified as EC 3.2.1.133 from Bacillus stearothermophilus followed by separating the maltose using ultra filtration process. Evaporation of the dilute permeate containing the maltose is expensive because of high energy cost and also faces a very high risk of microbial contamination.
Traditionally grains such as wheat, malt, sorghum (milo), millet (ragi), particularly whole grains are used in nutrition as carriers of macro- and micro-elements, proteins, fiber and vitamins. The majority of cereal grains appeared to be too readily digested to play an effective role as prebiotics or even as nutraceuticals. It has been suggested that designing genetically modified, less digestible cereals suitable as prebiotics to manipulate gut microflora (Gibson, G. R, and Roberfroid, M. B. 1995, Dietary modulation of the human colonic micrflora: Introducing the concept of prebiotics. J. Nutr. 125, 1401-1412).
There is a continuing interest in methods for producing grain compositions with isomalto-oligosaccharides enzymatically derived from the source substrate, e.g., grain or tuber, without having to separate the starch from other grain components and/or subject the starch of the substrate to high temperatures of jet cooking prior to transglucosidation action. There is also a continuing interest in low pH processes for minimizing the risk of microbial contamination. The present invention addresses these interests.